á551ñALPHA TOCOPHEROL ASSAY
The following procedure is provided for the determination of tocopherol as an ingredient.

Hydrogenator— Asuitable device for low-pressure hydrogenation may be assembled as follows.Arrange in a rack or in clamps two conical centrifuge tubes,connected in series by means of glass and inert plastic tubing and suitable stoppers of glass,polymer,or cork (avoiding all use of rubber).Use one tube for the blank and the other for the assay specimen.Arrange a gas-dispersion tube so that the hydrogen issues as bubbles at the bottom of each tube.Pass the hydrogen first through the blank tube and then through the specimen tube.

Procedure— Pipet into a suitable vessel 25mLof the final washed ether solution of the unsaponifiable fraction obtained as directed for When Tocopherol Is Presentunder Procedurein the Vitamin A Assay á571ñ,and evaporate to about 5mL.Without applying heat,remove the remaining ether in a stream of inert gas or by vacuum.Dissolve the residue in sufficient alcohol to give an expected concentration of about 0.15mg of alpha tocopherol per mL.Pipet 15mLinto a 50-mLcentrifuge tube,add about 200mg of palladium catalyst,stir with a glass rod,and hydrogenate for 10minutes in the Hydrogenator,using hydrogen that has been passed through alcohol in a blank tube.Add about 300mg of chromatographic siliceous earth,stir with a glass rod,and immediately centrifuge until the solution is clear.
Test a 1-mLaliquot of the solution by removing the solvent by evaporation,dissolving the residue in 1mLof chloroform,and adding 10mLof antimony trichloride TS:no detectable blue color appears.[NOTE—If a blue color appears,repeat the hydrogenation for a longer time period,or with a new lot of catalyst.]
Pipet 2mLof the supernatant into a glass-stoppered,opaque flask,add 1.0mLof a 1in 500solution of ferric chloride in dehydrated alcohol,*and begin timing the reaction,preferably with a stop watch.Add immediately 1.0mLof a 1in 200solution of 2,2¢-bipyridine in dehydrated alcohol,mix with swirling,add 21.0mLof dehydrated alcohol,close the tube,and shake vigorously to ensure complete mixing.When about 9½minutes have elapsed from the beginning of the reaction,transfer part of the mixture to one of a pair of matched 1-cm spectrophotometer cells.After 10minutes,accurately timed,following the addition of the ferric chloride-dehydrated alcohol solution,determine the absorbance at 520nm,with a suitable spectrophotometer,using dehydrated alcohol as the blank.Perform a blank determination with the same quantities of the same reagents and in the same manner,but using 2mLof dehydrated alcohol in place of the 2mLof the hydrogenated solution.Subtract the absorbance determined for the blank from that determined for the assay specimen,and designate the difference as AD.
Calculate the alpha tocopherol content,in mg,in the assay specimen taken by the formula:
30.2AD/(LCD),
in which ADis the corrected absorbance;Lis the length,in cm,of the absorption cell;and CDis the content of the assay specimen in the alcohol solution employed for the measurement of absorbance,expressed as g,capsules,or tablets per 100mL.
*  NOTE—The absorbance of the blank may be reduced,and the precision of the determination thereby improved,by purification of the dehydrated alcohol that is used throughout the assay.Purification may be accomplished by the addition of a few crystals (about 0.02%)of potassium permanganate and of a few pellets of potassium hydroxide to the dehydrated alcohol,and subsequent redistillation.